Confounders of mutation-rate estimators: selection and phenotypic lag in Thermus thermophilus

Abstract

In a recent description of the rate and character of spontaneous mutation in the hyperthermophilic bacterium Thermus thermophilus, the mutation rate was observed to be substantially lower than seen in several mesophiles. Subsequently, a report appeared indicating that this bacterium maintains an average of about 4.5 genomes per cell. This number of genomes might result in a segregation lag for the expression of a recessive mutation and might therefore lead to an underestimate of the rate of mutation. Here we describe some kinds of problems that may arise when estimating mutation rates and outline ways to adjust the rates accordingly. The emphasis is mainly on differential rates of growth of mutants versus their parents and on various kinds of phenotypic lag. We then apply these methods to the T. thermophilus data and conclude that there is as yet no reliable impact on a previously described rate.

Keywords: Differential growth rates; Fluctuation test; Mutation rate; Phenotypic lag.

Fig. 1

Simulation of phenotypic lag in a bacterial population composed of cells maintaining four initially identical copies of their genome, typically as one circular chromosome per genome. In each generation, all copies replicated and each cell divided into two daughter cells, each receiving four chromosome copies. Mutations occurred randomly at a rate per gene copy per cell division of μ = 3.468 × 10^–7 (a value reflecting the T. thermophilus estimate) and genomes were allocated to each pair of daughter cells at random. The horizontal axis (g) shows the number of generations after the first mutation occurred in each simulated culture. Production of the first cell containing four mutant genomes (which is when the phenotype was first expressed in at least one progeny of the first-mutated cell) followed within a few generations and continued cell division was simulated for at least nine generations after that time. The numbers of cells in the mutant lineage containing 0, 1, 2, 3, or 4 mutant chromosomes were tabulated and expressed as a function of g. The entire simulation was repeated 200 times. The solid curve is the fraction of mutations that were expressed as predicted by the expression (g – L)/g in section 2.2. At each generation, the top and bottom vertical dashed lines mark 1.5 interquartile ranges outside of the 75^th and 25^th percentiles, respectively, and values indicated by dots are considered to be outliers. The boxes indicate the middle 50% of values and the horizontal bars within the boxes mark the medians. The median value at g = 3 is based on tiny numbers of mutants and is correspondingly unreliable, that it, is itself an outlier that should be ignored.